The subcutaneous (SQ) administration of insulin to IDDM patients has saved millions of lives, however the serious circulatory and organ failure related complications of IDDM are still a major cause of disability and death in the US. These sequela are a direct result of hyper- and hypoglycemia associated with the delayed therapeutic response of current insulin formulations. The major deficiency in current administration of insulin is its failure to accurately model the rapid and exact release of insulin that is achieved by a healthy pancreas. The ideal insulin should be fast acting after SQ injection, resulting in rapid reduction of glucose levels, provide basal physiological levels of insulin, be non-immunogenic, and be protected from enzymatic degradation. Commercially available insulin is a fairly unstable preparation, and care must be exercised by the patient to prevent extremes in temperature and excessive agitation/shaking, which can lead to aggregation and precipitation of the protein, diminishing the therapeutically available dose. In addition, SQ insulin administration results in additional insulin aggregation, preventing fast absorption kinetics and rapid biological activity. Other problems associated with current insulin preparations include enzymatic degradation of the protein in the SQ injection site and immunogenicity towards xenograph insulin preparation (porcine, bovine). In this application, new PEG-insulin conjugates will be synthesized by chemically linking human insulin with poly(ethylene glycol) (PEG) through the amino groups on insulin (Gly A1, Phe B1, and LysB29). Previously, the investigators have chemically modified insulin and established that site specific amino group modification will alter the biological activity of the protein. Furthermore, PEG is available in a wide range of MW, each providing different physical characteristics to modified proteins. Thus, variables to be explored in optimizing the PEG-insulin conjugate will be the MW of PEG and the site specific attachment of PEG. This laboratory has over two decades of experience involving research of biomedical polymers, therapeutics, and drug delivery. Through preliminary data, the hypothesis of this proposal has been partly proven: PEG-insulin conjugates were synthesized and characterized, resulting in improved stability and biological activity. Thus, by expanding the variables of PEG-insulin conjugates (MW and site specific modification), we can optimize the therapeutic effectiveness to provide a fast acting, longer basal effect, and non-immunogenic PEG-insulin conjugate. This proposal will focus on the synthesis and optimization of PEG-insulin conjugates. The bioactivity, enzyme degradation, immunogenicity and pharmacokinetic properties of the optimized PEG-insulin conjugate will be determined. The biological effectiveness of the PEG-insulin conjugates will also be investigated in approved animal models to optimize and correlate the variable chemical modification with bioactivity. After long-term animal studies, optimized PEG-insulin will be selected for further clinical applications.